Effect Of MyD88 In Maturation Of Dendritic Cells Stimulated By OK-432

Objective To approach the effects of myeloid differentiation factor 88(MyD88) siRNA on the function dendritic cells(DCs) from mouse bone marrow stimulated by OK-432, and offer a new idea basis of the maturation mechanism on DCs.Methods Mouse bone marrow monocytes(BMMCs) were isolated by adherence method and cultured in RPMI1640 containing 10% FCS, rmGM-CSF and rmIL-4 and then stimulated by OK-432 so as to control the maturation of DCs in vitro . DCs were divided into blank control group, OK-432 group and RNA interference group. Control group was added nothing, and OK-432 group was added OK-432 at the final concentration 5μg/ml. MyD88 siRNA was added in RNA interference group, and 12 hours later OK-432 at the final concentration of 5μg/ml was added. All groups were cultured for 48 hours thereafter. Cells were observed by the inverted microscope and Wright-Giemsa stain. Method of flow cytometry were used to determined cells surface antigens. RT-PCR was applied to measure the expression of MyD88 and NF-кB mRNA. ELISA was used to detect the concentration of IL-12 and TNF-αin the supernatant. Antigen presenting ability of DCs in allo-MLR and cytotoxic effect were measured by MTT assay .Results MyD88 and NF-кB was high expression , TNF-αand IL-12 in concentration the supernatant were increased and the allo-stimulate capacity and cytotoxic effect of DCs were enhanced by treating with OK-432 .But MyD88 siRNA can inhibit all these responses.Conclusion Mouse bone marrow monocytes(BMMCs) were isolated by adherence method and induced by rmGM-CSF combined with rmIL-4 and then were impulsed with OK-432,which can abundantly amplify maturation DCs.The target MyD88 siRNA can reduce MyD88 expression in mouse myeloid DCs and inhibit the maturation of DCs stimulated by OK-432,it indicates that OK-432 induce the maturation of DCs mediated by MyD88.